The molecular events involved in the establishment of anti-viral and cytostatic effects of interferon are poorly understood. We have observed that a line of mouse cells deficient in the enzyme thymidine kinase (Ltk- cells) fail to respond to interferon by induction of an anti-viral state or synthesis of double-stranded RNA-dependent enzymes. Clones derived from the Ltk-cells by introduction of the Herpes virus thymidine kinase gene and selection in HAT-medium, were responsive to interferon in terms of both anti-viral activity and induction of specific enzymes. We propose here to use similar procedures to establish whether acquisition of interferon sensitivity is due to the uptake and expression of thymidine kinase genes. Clones derived by DNA-mediated transfer of various thymidine kinase genes in the absence and presence of carrier DNA and of the adenine phosphoribosyl transferase gene will be treated with interferon and viral cytotoxicity, inhibition of viral protein synthesis and induction of double-stranded RNA-dependent enzymes will be monitored. The interferon sensitivity of other thymidine kinase deficient mouse and human cells will also be tested. Human cells lacking both tk and sensitivity to interferon (A-673 cells) will be used to generate clonal lines expressing the Herpes virus tk gene. Interferon sensitivity will be studied by measuring resistance to viral infection and induction of double-stranded RNA-dependent enzymes. Such human tk(+) cells will be cultured in bromodeoxyuridine to produce revertant sub-clones which have lost the tk gene and a correlation between interferon sensitivity and the presence of functional tk genes sought. In order to determine whether responses to interferon other than induction of an anti-viral state require thymidine kinase, the effect of interferon on cell growth and DNA synthesis will be studied in murine and human tk(-) and tk(+) cells. By establishing whether or not thymidine kinase is involved in the activation of responses to interferon in mouse and/or human cells we hope to open a new area for studying the mechanism of interferon action.